HAL Id: hal-03006246
https://hal.archives-ouvertes.fr/hal-03006246
Submitted on 15 Nov 2020
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of
sci-entific research documents, whether they are
pub-lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
methylation and accessibility, histone marks, and
hTERT gene expression in acute promyelocytic leukemia
cells: perspectives for all- trans retinoic acid in cancer
therapy
Delphine Garsuault, Claire Bouyer, Eric Nguyen, Rohan Kandhari, Martina
Prochazkova-carlotti, Edith Chevret, Patricia Forgez, Evelyne
Ségal-bendirdjian
To cite this version:
Delphine Garsuault, Claire Bouyer, Eric Nguyen, Rohan Kandhari, Martina Prochazkova-carlotti, et
al.. Complex context relationships between DNA methylation and accessibility, histone marks, and
hTERT gene expression in acute promyelocytic leukemia cells: perspectives for all- trans retinoic acid
in cancer therapy. Molecular Oncology, Elsevier, 2020, 14, pp.1310 - 1326. �10.1002/1878-0261.12681�.
�hal-03006246�
and accessibility, histone marks, and
hTERT gene
expression in acute promyelocytic leukemia cells:
perspectives for all-
trans retinoic acid in cancer therapy
Delphine Garsuault1,2,3, Claire Bouyer1,2, Eric Nguyen1,2, Rohan Kandhari1,4, Martina
Prochazkova-Carlotti5, Edith Chevret5, Patricia Forgez1,2and Evelyne Segal-Bendirdjian1,2,6
1 Team: Cellular Homeostasis, Cancer, and Therapies, INSERM UMR-S 1124, Universite de Paris, France 2 Universite de Paris, Paris Sorbonne Cite, France
3 Paris-Sud University, Paris-Saclay University, Orsay, France 4 Indian Institute of Technology, BHU, Varanasi, India
5 Team Cutaneous Lymphoma Oncogenesis, INSERM U1053, Bordeaux, France 6 BioMedTech Facilities, CNRS UMS2009/INSERM US36, Universite de Paris, France
Keywords
acute promyelocytic leukemia; ATRA; DNA methylation; histone marks;hTERT promoter; telomerase
Correspondence
E. Segal-Bendirdjian, Team: Cellular Homeostasis, Cancer, and Therapies, INSERM UMR-S 1124, Universite de Paris, 45 rue des Saints-Peres, Paris, France Fax: +33 1 42 86 21 62
Tel: +33 1 42 86 22 46
E-mail: evelyne.segal-bendirdjian@inserm.fr (Received 24 December 2019, revised 19 February 2020, accepted 28 March 2020) doi:10.1002/1878-0261.12681
Telomerase (hTERT) reactivation and sustained expression is a key event in the process of cellular transformation. Therefore, the identification of the mechanisms regulating hTERT expression is of great interest for the development of new anticancer therapies. Although the epigenetic state of
hTERTgene promoter is important, we still lack a clear understanding of
the mechanisms by which epigenetic changes affect hTERT expression. Retinoids are well-known inducers of granulocytic maturation in acute promyelocytic leukemia (APL). We have previously shown that retinoids repressed hTERT expression in the absence of maturation leading to growth arrest and cell death. Exploring the mechanisms of this repression, we showed that transcription factor binding was dependent on the epige-netic status of hTERT promoter. In the present study, we used APL cells lines and publicly available datasets from APL patients to further investi-gate the integrated epigenetic events that promote hTERT promoter transi-tion from its silent to its active state, and inversely. We showed, in APL patients, that the methylation of the distal domain of hTERT core pro-moter was altered and correlated with the outcome of the disease. Further studies combining complementary approaches carried out on APL cell lines highlighted the significance of a domain outside the minimal promoter, localized around 5 kb upstream from the transcription start site, in activat-ing hTERT. This domain is characterized by DNA hypomethylation and H3K4Me3 deposition. Our findings suggest a cooperative interplay between
hTERTpromoter methylation, chromatin accessibility, and histone
modifi-cations that force the revisiting of previously proposed concepts regarding
hTERT epigenetic regulation. They represent, therefore, a major advance
in predicting sensitivity to retinoid-induced hTERT repression and, more
Abbreviations
APL, acute promyelocytic leukemia; ATO, arsenic trioxide; ATRA, all-trans retinoic acid; BAC, bacterial artificial chromosome; FISH, fluorescencein situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hTERT, human telomerase reverse transcriptase; hTR, human telomerase RNA; NOMe-seq, nucleosome occupancy and methylome sequencing; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; RRBS, reduced representation bisulfite sequencing; SAM,S-adenosylmethionine; SNP, single nucleotide polymorphism; SSC, saline sodium citrate; TPE-OLD, telomere position effect over long distances; TSS, transcription start site.
generally, in the potential development of therapies targeting hTERT expression in cancers.
1. Introduction
Telomere maintenance is a primary and universal char-acteristic of cellular transformation, leading to
unlim-ited replicative capacity. Without telomere
maintenance, the other hallmarks of cancer described
by Hanahan and Weinberg (2011) could neither persist
nor contribute to the subsequent cancerous events. This maintenance is performed mainly by a specialized ribonucleoprotein complex, the telomerase. Any strat-egy to block telomerase activity or expression specifi-cally in tumor cells, to force them to enter replicative senescence or apoptosis, may contribute to innovative therapeutic developments. Up to now, strategies tar-geting telomerase activity inhibition have been disap-pointing because of low efficacy and long-term toxicity of the drugs. These failures are partially due to an insufficient understanding of telomerase regulation mechanisms.
The human telomerase complex consists of a cat-alytic reverse transcriptase protein subunit (hTERT) encoded by TERT gene located on chromosome 5
(5p15.33) (Meyerson et al., 1997), an RNA template
(hTR) encoded by TERC gene located on chromosome
3 (3q26.2) (Shippen-Lentz and Blackburn, 1990) and
accessory proteins required for proper telomerase assembly and recruitment to chromosomes (Cohen
et al.,2007).
hTERT expression is the primary determinant and
the limiting factor for telomerase activity. The regula-tion of hTERT expression in human cancers is conse-quently of major importance. hTERT expression is tightly regulated at the transcriptional level (Avilion
et al., 1996). As the reactivation of hTERT is critical
in carcinogenesis and tumor progression, it is essential to further advance in our understanding of hTERT regulation at the transcriptional level. Several tran-scription factors, either repressors (such as Mad1, E2F, WT1, and MZF2) or activators (such as c-Myc, NF-kB, and Sp1) are important in the tight control of
hTERTexpression (Ramlee et al.,2016; Renaud et al.,
2005). However, these factors are involved in the
regu-lation of numerous normal cells and thereby are diffi-cult to be specifically targeted in cancer cells. Recent studies have identified cancer-associated hTERT pro-moter mutations as a genetic mechanism for hTERT upregulation. The most frequent mutations are found
upstream of the transcription start site (TSS) at 1 295 228 (C288T), and 1 295 250 (C250T). These mutations generate novel binding sites for the ETS (E26 transformation-specific or E-twenty-six) tion factors and thereby, alter positively the
transcrip-tional regulation of hTERT (Bell et al., 2015; Horn
et al., 2013; Huang et al., 2013; Vinagre et al., 2013).
Besides, hTERT upregulation occurs in the absence of these promoter mutations in many tumor types, sug-gesting that other mechanisms are involved, and in particular, epigenetic mechanisms. Indeed, the epige-netic state of hTERT promoter is important for the tight control of hTERT expression. However, despite extensive studies on hTERT promoter DNA
methyla-tion alteration, contradicting results have been
reported in the literature (Azouz et al., 2010; Dessain
et al., 2000; Devereux et al., 1999; Guilleret and
Ben-hattar, 2003; Guilleret et al., 2002; Losi et al., 2019;
Zinn et al., 2007). Therefore, we still lack a clear
understanding of the underlying mechanisms by which epigenetic changes affect hTERT expression, and if they can be specifically targeted.
DNA methylation is often linked to histone post-translational modifications (Bannister and Kouzarides,
2011) that affect the compaction state of chromatin,
and thereby gene expression by controlling the accessi-bility of transcription factors to the promoter. Nucleo-somes have classically been thought to prevent DNA sequence from interacting with transcription factors (either activators or repressors). Therefore, the degree of nucleosome occupancy along DNA in the chro-matin contributes significantly in the activation and repression of chromatin regions because it modulates the accessibility of DNA to the transcriptional
machin-ery and regulatory proteins (Li et al.,2007). Many
fac-tors have been proposed to directly regulate the
nucleosome positioning (Lai and Pugh, 2017),
includ-ing the genomic sequence, DNA methylation
(Cho-davarapu et al., 2010), and histone modifications.
Importantly, the higher-order chromatin organization results in the formation of looping structures that could play a significant role in gene activity by cluster-ing genomic loci into domains via long-range
interac-tions (Rowley and Corces, 2018). This chromatin
looping is also influenced by nucleosome positioning and therefore by epigenetic modifications. The tight
methylation, histone marks, chromatin accessibility and subnuclear localization of hTERT has not been previously formally examined and was addressed in this study.
All-trans retinoic acid (ATRA) is widely used as first-line therapy in acute promyelocytic leukemia (APL) as an inducer of granulocytic maturation of APL blasts. Besides, we have previously reported that long-term ATRA treatment could induce telomere-dependent cell death in some ATRA-maturation-resistant cells. Indeed, in the maturation-resistant NB4-LR1 cells, pharmaco-logical concentrations of ATRA induced strong repres-sion of hTERT leading to telomere shortening and cell
death (Pendino et al.,2001,2003,2006). This
observa-tion suggests that ATRA, by targeting hTERT, can exert antitumoral properties independently of its action on differentiation. A variant of the NB4-LR1 cell line,
named NB4-LR1SFD, resistant to telomerase-dependent
ATRA-induced cell death was selected. The
NB4-LR1SFDcells are characterized by a steady expression of
hTERTdespite the continuous presence of ATRA
(Pen-dino et al., 2002). Understanding the mechanisms of
ATRA-induced hTERT repression will lead to the devel-opment of new therapeutical strategies to improve ATRA responsiveness. Thus, the two ATRA-matura-tion-resistant APL cell sublines described above, which behave distinctly to long-term ATRA treatment regard-ing the influence on hTERT expression, are a valuable model to study the molecular events leading to hTERT repression and reactivation in cancer. We have previ-ously shown that transcription factor binding to hTERT promoter is dependent on the epigenetic status of the promoter, including DNA methylation (Azouz et al.,
2010). In the present study, we took advantage of the
two well-established cell lines, in which hTERT expres-sion and telomerase activity are oppositely regulated by retinoids to explore in more details the interplay between hTERT gene positioning in the nucleus, DNA methylation, nucleosome occupancy, and histone modi-fications at hTERT promoter. To this end, our investi-gation combines a highly sensitive single-molecule nucleosome occupancy and methylome sequencing assay (NOMe-seq) with histone ChIP analysis and 3D-FISH to generate an integrated view of chromatin orga-nization and gene expression at the level of hTERT.
2. Materials and methods
2.1. Patients
DNA methylation data from bone marrow samples of 18 APL patients at diagnosis, eight matched patient at
remission, one sample from an APL patient, treated or
not ex vivo with ATRA for 48 h, CD34+cells from eight
healthy donors, and promyelocytes generated in vitro from these CD34 cells, were downloaded from the GEO
website (https://www.ncbi.nlm.nih.gov/gds/?term=
GSE42119[Accession]) (Schoofs et al., 2013). Histone
modifications from three APL patients (pz-302, a non-high-risk primary APL patient and pz-284 and pz-289, two high-risk primary APL patients resistant to stan-dard ATRA plus chemo treatment) were downloaded from the BLUEPRINT data portal (http://dcc.blue
print-epigenome.eu/#/experiment) and visualized in
UCSC genome browser (https://genome.ucsc.edu/).
2.2. Chemicals, cell lines, cell culture, and treatments
All-trans retinoic acid (ATRA), arsenic trioxide
(ATO), and protease inhibitor cocktail (P8340) were purchased from Sigma (St Louis, MO, USA). The maturation-resistant human APL cell lines, NB4-LR1
and NB4-LR1SFD, were cultured as previously
described (Pendino et al., 2001, 2003). All cells were
cultured at 37°C in a humidified incubator with 5%
CO2 (Binder Incubators, Nanterre, France). For
treat-ments, cells were seeded in medium containing 1µMof
ATRA, 0.2µMof ATO alone or in combination.
2.3. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Courtaboeuf, France) according to the manufacturer’s instructions and subjected to reverse transcriptase reaction with oligo(dT) using Transcriptor First Strand cDNA Syn-thesis kit (Roche Diagnostics, Meylan, France) as described in the manufacturer’s instructions. The cDNAs were subsequently submitted to quantitative real-time PCR using the LightCycler technology and the Light Cycler FastStart DNA MasterPLUS SYBR Green Kit (Roche Diagnostics). hTERT levels were nor-malized to the expression of glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) used as the internal control gene. Primer sequences and their localization on the hTERT gene are shown in Table S1 and Fig. S1.
2.4. Sanger sequencing
The presence of hTERT promoter/enhancer mutations was evaluated by conventional Sanger sequencing. Genomic DNA was extracted from cells as previously reported (Segal-Bendirdjian and Jacquemin-Sablon,
1995). hTERT core promoter (region I: from the
posi-tion650 to +150 bp relative to the TSS) and a distal
regulatory upstream region (region II: from the
posi-tion 5500 to 4900 bp relative to the TSS) were
amplified using specific primers whose sequences and localizations are reported in Table S1 and Fig. S1.
2.5. Nucleosome occupancy and methylome sequencing
Nucleosome occupancy and methylome sequencing was performed as previously described (Kelly et al.,
2012). In brief, nuclei were isolated, resuspended in
165µL ice-cold GpC buffer [19 M.CviPI GC buffer,
0.3M sucrose, 160µM S-adenosylmethionine (SAM)],
and split into two aliquots. One aliquot was treated with 75 U of GpC methyltransferase M.CviPI, the other was incubated with the same amount of water.
The tubes were incubated for 15 min at 37°C before
adding a boost of 75 U of M.CviPI, and 160µMSAM
in the treated nuclei for an additional 15 min at 37°C.
The reaction was terminated by the addition of one
volume of stop solution (20 nM Tris/HCl pH 8,
600 mM NaCl, 1% SDS, 10 mM EDTA, 400µgmL1
proteinase K) and the samples were incubated for 2 h
at 55°C. DNA was then purified by
phenol/chloro-form extraction and ethanol precipitation. Bisulfite
conversion was performed on 1µg of purified DNA
using the EZ DNA Methylation Kit (Zymo Research, Ozyme, SAS, Saint-Cyr-L’Ecole, France). Bisulfite-converted DNA was used for PCR amplification of the regions of interest with the primers reported in Table S1. We designed NOMe-seq assays to explore region I and II as defined above (see Fig. S1). PCR amplicons were purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Hoerdt, France) and cloned into the pGEM-T Easy vector (Promega, Charbonnieres-les-Bains, France) as described in the
manufacturer’s instructions. For each experiment, 10–
20 plasmid subclones were sequenced (MWG Biotech, Ebersberg, Germany) for the assessment of nucleo-some occupancy and CpG methylation. The M.CviPI enzyme methylates GpC sites in accessible DNA, whereas nucleosome bound DNA, which is inaccessi-ble, remains refractory to GpC methylation. Reactions without M.CviPI were routinely performed to confirm endogenous CpG methylation levels. Besides, NOMe-seq retains the endogenous methylation status of the DNA allowing nucleosome positions and DNA methy-lation to be determined on the same molecule. The efficiency of M.CviPI GpC methyltransferase was high (95%) and the bisulfite conversion rate was 99% on average. The methylation patterns of the individual
clones are presented in Fig. S2. The data visualization as methylation profiling plot was performed using
Methylation plotter web tool (Mallona et al.,2014).
2.6. Chromatin immunoprecipitation
ChIP assay was performed using Magna ChIP kit (Merck Millipore, Guyancourt, France), following the
manufacturer’s instructions. Briefly, 107 cells were
cross-linked with 1% paraformaldehyde and sonicated to obtain fragments ranging from 300 to 600 bp (Bioruptor Pico; Diagenode Diagnostics, Seraing,
Bel-gium). An aliquot (3 µL, 6000 cells) of the sonicated
chromatin was used as input fraction to quantify the
total amount of DNA. For immunoprecipitation, 4 µg
of antibodies were prebound to 20 µL protein A/G
magnetic beads and incubated with chromatin
over-night at 4 °C. As a negative control, IgG of the same
species as the antibody of interest was included. The following antibodies were used for the
immunoprecipi-tation: H3K27Me3 (Merck Millipore, #07-449);
H3K4Me3 (Merck Millipore, #04-745); H3Ac (Merck Millipore, #06-599), H3K9Me3 (Merck Millipore, #07-442); IgG (Merck Millipore, #PP64B). Three biological replicates were produced independently at a different time of cell culture. Immunoprecipitated chromatin samples were un-cross-linked and purified. Immuno-precipitated and input DNA were then analyzed by quantitative polymerase chain reaction (qPCR, Light-Cycler 2.0; Roche) with the appropriate primers target-ing regions upstream and downstream hTERT TSS (Fig. S1). The primer sequences are indicated in Table S1. The amount of immunoprecipitated DNA is represented as a normalized signal to total input DNA used in each immunoprecipitation.
2.7. 3D fluorescence in situ hybridization
Fluorescence in situ hybridization assay was performed
as previously described (Chaumeil et al.,2013). Briefly,
2 9 106cells were fixed with 2% paraformaldehyde and
spread on Superfrost slides (Menzel-Gl€azer, Fisher Sci-entific, Illkirch, France) before permeabilization with 0.5% Triton in PBS. Slides were dehydrated with a gradual concentration of ethanol (70%, 85%, and
100%) and then treated with RNase A (100 µgmL1)
for 1 h at 37 °C (Neobrite, NB12-0001;
NeoBiotech, Rotterdam, Netherlands). A second
per-meabilization step with 0.7% Triton in 0.1MHCl was
performed before denaturation with 50% formamide in
29 SSC (saline sodium citrate) solution at 80 °C for
30 min. Probes were denatured at 75°C for 5 min and
37°C. After 3 washes in 29 SSC/50% formamide, three washes in 29 SSC, one wash in 0.59 SSC and one wash in PBS, slides were incubated with Hoechst 33342
(1µgmL1 in PBS) for 15 min at room temperature
and mounted with Mowiol. Probes were prepared using nick translation kit (Vysis kit; Abbott Laboratories, Rungis, France) following the manufacturer’s instruc-tions. Bacterial artificial chromosome (BAC) plasmids were purchased from Source BioScience (Biovalley SA, Illkirch, France: RP11-117B23 for hTERT locus stain-ing (Spectrum Orange, 552 nm), RP11-44H14 for sub-telomeric region 5p staining (Spectrum Green, 496 nm), and RP11-846K3 for staining ‘intermediary’ region, containing TPPP and CEP72 genes (Spectrum Green, 496 nm). The positions of the FISH probes are indi-cated in Fig. S2. Labeled probes were precipitated by adding 10-fold excess of Cot-1 DNA, 1/10 volume of
3M sodium acetate (NaAc, pH 5.2) and 3 volumes of
100% ethanol. Probes were then centrifuged at 12 000 g
for 45 min at 4°C. After washing with 70% ethanol,
probes were then dried at 37°C for 10 min and
resus-pended in 50µL of hybridization buffer (29 SSC/50%
formamide/10% dextran sulfate). Images were acquired using LSM 710 confocal microscope (Carl Zeiss, Marly
le Roi, France). A 639 Plan-Apochromat oil immersion
objective was used to capture optical sections at
inter-vals of 0.37µm. LSM-type images were generated and
processed with IMAGEJ (https://imagej.nih.gov/ij/) and
JACoP plugin. The 3D distance between each center of the deconvolved fluorescent spot of hTERT locus, and either the subtelomeric 5p region or the intermediate
locus were collected and analyzed with GRAPHPAD
PRISM(San Diego, CA, USA).
2.8. Statistical analysis
Statistical analysis was conducted using GRAPHPAD
PRISM 6.01 software. The difference between groups
was analyzed using unpaired or paired Student’s t-test when there were only two groups or assessed by one-way ANOVA followed by Tukey’s multiple compar-ison tests when there were more than two groups. All tests carried out were two-tailed. Differences were
con-sidered as significant when P< 0.05.
3. Results
3.1. Identification of anhTERT promoter
methylation signature in APL patient samples In a previous work performed on APL cell lines, DNA methylation analysis of the hTERT promoter
led us to identify two distinct functional domains dif-ferentially methylated, a proximal one fully
unmethy-lated and a distal one whose methylation
modifications could account for the capacity of
ATRA to repress hTERT gene (Azouz et al., 2010).
To evaluate the clinical relevance of this particular epigenetic pattern, we analyzed the methylation
pro-file of hTERT promoter up to 5 kb upstream the
TSS in eighteen patient samples at primary diagnosis using a publicly available APL dataset (Schoofs
et al., 2013) (GSE42119). Bone marrow samples of
eight matched patients in remission were also
ana-lyzed. CD34+ cells from healthy donors and
promye-locytes generated in vitro from these CD34+ cells were
included in the analysis as controls. Raw methylation levels from CpG sites covered by RRBS across all samples were used to perform unsupervised
hierarchi-cal clustering (Fig.1A). The samples formed two
main clusters: one (top) encompassing APL patient samples at diagnosis and one (bottom) encompassing bone marrow samples of patients in remission and hematopoietic progenitor cells from healthy donors. In patients at diagnosis, we identified a region close to the TSS that was largely hypomethylated, while
the distal region further upstream (200 bp upstream
of the TSS) was significantly more methylated. The dual methylation pattern, already reported in APL
cell lines (Azouz et al., 2010), was therefore
con-firmed in APL patient samples at diagnosis. Interest-ingly, the global methylation level of the distal region of the core promoter differs and this difference clus-ters with the patient conditions. Indeed, the methyla-tion of the CpG sites within the distal region was significantly lower in patients at remission and in healthy donors compared to patients at diagnosis. Only one patient at diagnosis clusters with the remis-sion patients. It is worth noting that in vitro ATRA treatment of primary APL cells from one APL patient for 48 h did not change significantly the
pat-tern of DNA methylation of hTERT promoter
(Fig.1B). One potential explanation for this might be
that a 48-h ATRA treatment of APL cells in vitro could not completely mimic conditions of ATRA treatment of patients. Of note, no expression data of
hTERTrelative to the dataset analyzed were available
neither for APL patients at diagnosis nor for patients at remission. This rules out the possibility of per-forming any correlation between the methylation pat-tern of hTERT gene promoter and its expression.
Nevertheless, these results underline the significance of the epigenetic modification of this distal region in
hTERT expression regulation, and indicate that the
a potential indicator for the diagnosis and the moni-toring of APL disease outcome.
3.2. NB4-LR1 and NB4-LR1SFD, two
ATRA-maturation-resistant APL cell lines, a tool to investigate the epigenetic regulation of the hTERT gene
The results obtained in APL patients encouraged us to perform a more comprehensive analysis of the epige-netic status of hTERT gene promoter. To carry out this study, we took advantage of two well-established APL cell lines, in which hTERT expression and telom-erase activity are regulated by retinoids in an opposite way. The NB4-LR1 cell line, derived from an APL patient, is resistant to ATRA-induced maturation
(Duprez et al., 1992; Lanotte et al., 1991). In this cell
line, as previously reported, the long-term treatment
with ATRA induced a strong repression of hTERT
(Fig. 2). In the NB4-LR1SFDcell line, established from
the NB4-LR1 cells, hTERT expression has been stably
reactivated (Pendino et al., 2001, 2002). In this cell
line, the constitutive expression of hTERT is higher than in the NB4-LR1 cell line and remained high despite ATRA treatment. hTERT repression is, how-ever, achieved when ATRA treatment was combined
with ATO (Tarkanyi et al.,2005). Therefore, these two
specific cell lines are illustrative of tumor progression process, and they represent excellent tools to under-stand how tumor cells can install a finely tuned tran-scriptional regulation for hTERT and bypass its repression. One mechanism that can explain, at least partly, the reactivation of hTERT is mutations at specific loci of the hTERT promoter (Vinagre et al.,
2013). hTERT promoter sequencing showed that
nei-ther NB4-LR1 nor NB4-LR1SFD cells harbored the
diagnosis remission APL patients Healthy donors promyelocytes CD34 Methylation (%) 0 20 40 60 80 100 Sample type
Enhancer Core promoter
TERT TSS
II
I
MZF2 MZF2 WT1 E boxSp1Sp1Sp1E box proximal distal diagnosis untreated (48h) ATRA treated (48h)In vitro treatment of an APL patient sample A
B
Fig. 1. Hierarchic cluster analysis of APL patients’ samples and methylation profile athTERT promoter and enhancer. (A) The methylation heat map generated from the unsupervised hierarchical clustering is based on raw RRBS (reduced representation bisulfite sequencing) DNA methylation values in patients at diagnosis and remission, and healthy donors. (B)In vitro ATRA treatment of one APL patient sample for 48 h. White color indicates unavailability of data. The color grid on each side visualizes the sample characteristics.
recurrent promoter mutations known to generate ETS
binding sites at 124 bp (C228T) and 146 bp
(C250T) upstream of the start codon (Bell et al.,2015;
Horn et al., 2013; Huang et al., 2013; Vinagre et al.,
2013). Sequence analysis revealed, the presence of
already known single nucleotide polymorphisms
(SNPs) in hTERT core promoter and a region approxi-mately 5 kb upstream of the hTERT TSS previously described as an enhancer element (Eldholm et al.,
2014) (Fig. S1 and Table S2). As the SNP observed
were found in both cell lines, they could not explain
the distinct responses of NB4-LR1 and NB4-LR1SFD
cells to long-term ATRA treatment regarding hTERT expression, indicating that other mechanisms may be involved, most likely epigenetic mechanisms. There-fore, the above cell lines would serve as valuable cell models to investigate epigenetic events involved in telomerase regulation.
3.3. hTERT promoter DNA methylation and
nucleosome occupancy by NOMe-seq in ATRA-maturation-resistant APL cell lines
To investigate the relationship between DNA methyla-tion and nucleosome occupancy in ATRA-treated and
untreated NB4-LR1 and NB4-LR1SFD cells and
resolve the pattern of methylation at hTERT promoter
gene, we applied a high-resolution, single-molecule analysis named NOMe-seq. This procedure allows the simultaneous investigation of nucleosome occupancy and endogenous CpG methylation on the same DNA molecule and the analysis of the relationships between these two chromatin features on a single locus (Kelly
et al., 2012). We focused on two different regions of
hTERT promoter: the first region (region I) extended
from 650 to +150 bp relative to TSS; the second
region (region II) located far upstream from the TSS
(5500 to 4900 bp) (Eldholm et al., 2014) identified
as a putative enhancer domain (Fig.3, Figs S1 and
S3). In region I, hTERT promoter DNA was weakly methylated with a mean methylation level of 23.7%
and 14.7% in untreated NB4-LR1 and NB4-LR1SFD
cells, respectively. We confirmed the dual methylation pattern observed in APL patients, as the region close to the TSS is largely hypomethylated in both cell lines,
while the region further upstream (about 600 to
200 bp upstream of the TSS) was significantly more
methylated (Zinn et al., 2007). In NB4-LR1 cells,
ATRA-induced repression of hTERT was associated with a global decrease of CpG methylation level in the region I (from 23.7% to 9.4%), suggesting that the methylation status of the proximal promoter may con-tribute to hTERT gene silencing. Associated with this decrease of DNA methylation, we observed a striking loss of chromatin accessibility particularly in the
region between 200 and +1 bp. The size of this
region is large enough to accommodate at least one nucleosome. This observation was supported by the enrichment of histone H3 observed in this region in
the ChIP-qPCR assay (Fig.4, panel 4, amplicon e).
Similarly, in NB4-LR1SFD cells, the important
repres-sion of hTERT after treatment with ATRA and ATO in combination was associated with a hypomethylation of region I (from 14.7% to 2.2%) and a global reduc-tion in chromatin accessibility. Of note, ATRA treat-ment alone did not affect either CpG methylation or
chromatin accessibility in those cells. In NB4-LR1SFD
cells, ATO treatment alone induced a decrease of DNA methylation only in the distal part of hTERT core promoter without major modifications in chro-matin accessibility.
In region II, NB4-LR1 cells displayed a marked glo-bal hypermethylation (89.1%). Despite no significant change in the DNA methylation profile of this region was observed in ATRA-treated NB4-LR1 cells, a decrease of chromatin accessibility was noticed. In
NB4-LR1SFD cells, this region displayed strikingly a
variable but globally lower methylation levels (33.5%) compared to that in NB4-LR1 cells. An increase of the DNA methylation associated with a partial increase in
NB4-LR1
NB4-LR1
SFD ATRA ATO–
+
–
–
–
–
–
–
+
+
+
+
h T ER T/ GA PD H( % c ont r o l) 0 5 0 1 0 0 1 5 0 2 0 0 * * * * * * * * * * * * * * * *Fig. 2.hTERT mRNA expression in ATRA-maturation-resistant APL cell lines. Cells were treated for 7 days with ATRA (1µM) alone or in combination with ATO (0.2µM).hTERT gene expression levels in
NB4-LR1 and NB4-LR1SFDcells were measured by qRT-PCR. The
levels were normalized to GAPDH expression and the results were expressed as a percentage of that detected in untreated NB4-LR1 cells (SEM). t-Test or one-way ANOVA with post hoc Tukey, *P < 0.05, ***P < 0.001, ****P < 0.0001.
chromatin accessibility is observed after ATRA
treat-ment of NB4-LR1SFD cells. However, neither DNA
methylation nor chromatin accessibility was modified after treatment with ATRA and ATO alone or in com-bination.
Altogether, these results indicate that changes in the methylation status of hTERT promoter are probably a necessary but not a sufficient condition for an efficient transcriptional repression of this gene; they need to be associated with a decrease in chromatin accessibility.
3.4. Histone marks athTERT promoter in
ATRA-maturation-resistant APL cell lines and patients The histone modifications have been reported to play a significant role in the regulation of gene expression,
including hTERT (Cong and Bacchetti, 2000; Lewis
and Tollefsbol, 2016; Won et al.,2002). Therefore, we
conducted site-specific ChIP-qPCR assays to examine the relationship between hTERT expression and his-tone marks at specific positions along the hTERT
gene. Antibodies specific to one repressive (H3
trimethylated lysine 27, H3K27Me3), two active (H3 trimethylated lysine 4, H3K4Me3, and acetylated lysine H3, H3Ac) marks, and the total histone H3
were used for ChIP assay (Fig. 4).
In both untreated cell lines, the active marks,
H3K4Me3 and H3Ac (Fig.4, panel 1 and 2), were
enriched at hTERT core promoter (region in gray mapped by primer sets d, and e), indicating a permis-sive transcriptional status of the chromatin. In NB4-LR1 untreated cells, the active H3K4Me3 mark
(Fig. 4, panel 2) was included within a larger region of
H3K27Me3 repressive mark (Fig. 4, panel 3). This
TSS CpG m ethy la ti on (% ) Gp C me th yl a ti o n (% ) (a c c es s ibi lli ty ) NO M e -se q MZF2 W T1E-boxSp1E-boxCTCF * * ** ** *** * * ** ******* ** ** 5100 5200 5300 5400 –5500– – – – –5000– – – – – – – * ** * ******* * 1 600 500 400 300 200 100 +1 +100 0.8 0.6 0.4 0.2 0 0.8 0.6 0.4 0.2 0 * * * * * ** 1 0.8 0.6 0.4 0.2 0 0.8 0.6 0.4 0.2 0 * MZF2 WT1E-boxSp1E-boxCTCF * **** * *** ******** * MZF2 WT1 E-boxSp1E-boxCTCF +1 +100 -100 -200 -300 -400 -500 * * * * * * ******* * -600 * * -4900 -5100 -5200 -5300 -5400 -5500 -5000 * 1 0.8 0.6 0.4 0.2 0 0.8 0.6 0.4 0.2 0 CpG me th yl a ti o n (% ) Gp C m e th yla ti o n (% ) (a c ce ssib ill it y) NO M e -s e q * **** * * 1 0.8 0.6 0.4 0.2 0 0.8 0.6 0.4 0.2 0 * ** ** * * * * * MZF2 WT1E-boxSp1E-boxCTCF TSS A NB4-LR1 B NB4-LR1SFD D NB4-LR1SFD C NB4-LR1SFD Sp1 Sp1 RARa-RXR GABPa Sp1
Sp1 RARa-RXR GABPa Sp1 RARa-RXRSp1GABPa Sp1 Sp1 RARa-RXR GABPa Non treated ATRA+ ATO Non treated ATRA Non treated ATRA Non treated ATO II I II I 4900 5100 5200 5300 5400 –5500– – – – –5000–4900 –600–500–400–300–200 –100 +1 +100 –5500–5400–5300–5200–5100–5000–4900 –600–500–400–300–200–100 +1 +100 5100 5200 5300 5400 –5500– – – – –5000–4900 –600–500–400–300–200 –100 +1 +100 5100 5200 5300 5400 –5500– – – – –5000–4900 –600–500–400–300–200–100 +1 +100 5100 5200 5300 5400 –5500– – – – –5000–4900 –600–500–400–300–200–100 +1 +100 5100 5200 5300 5400 –5500– – – – –5000– – – – – – – 1 4900 600 500 400 300 200 100 +1 +100
Fig. 3. Chromatin accessibility and endogenous CpG methylation at the hTERT gene promoter as determined by NOMe-seq analysis. NOMe-seq was used to determine in the same time the level of endogenous DNA methylation and chromatin accessibility on individual DNA molecules for two subregions of thehTERT gene promoter visualized on the upper part of the figure as region I and region II. Region I encompasseshTERT TSS, and region II corresponds to the upstream conserved sequence described as an enhancer. For each sequence, 10–20 clones were analyzed from DNA obtained from NB4-LR1 (panel A) and NB4-LR1SFD(panel B–D) cells treated or not as indicated. The upper part of each panel indicates endogenous DNA methylation, the lower part chromatin accessibility. Data visualization as methylation profiling plot was performed using Methylation plotter web tool (Mallonaet al.,2014). Each line represents for each group of samples the methylation mean for each position. Asterisks indicate a statistical significance between the treated and untreated groups as calculated by Kruskal–Wallis test (P < 0.05). Ticks in x-axis indicate individual CpG (upper panel) and GpC (lower panel), respectively. Transcription factor binding sites (colored boxes) and TSS (solid arrow) are depicted.
concurrence of H3K4Me3 and H3K27Me3 across the
hTERT promoter and TSS was already described
(Zinn et al., 2007). This epigenetic feature is a
charac-teristic of bivalent domains (or ‘poised’ promoters)
(Bernstein et al.,2006), suggesting an effective
plastic-ity of the chromatin at this location of the hTERT promoter, crucial for the transition between active and repressive states. This feature was not observed in
NB4-LR1SFD cells since H3K27Me3 remained very
low across the region of the hTERT gene probed by
the nine primer sets (Fig.4, panel 3). Remarkably, in
the region mapped by the primer set a (region in pink), H3K4Me3 and to a lesser extent H3Ac, were
significantly enriched in NB4-LR1SFD cells as
com-pared to NB4-LR1 cells (Fig.4, panel 2).
After a 7-day ATRA treatment, the levels of these active marks decreased dramatically only in NB4-LR1
cells and remained unchanged in NB4-LR1SFD cells.
Nevertheless, their presence decreased in NB4-LR1SFD
exposed to the combination of ATRA and ATO. The
1 2 3 4 5 6 7 8 9 10 11 12 13 141516 Exons a b f g h i d e ChIP c d e a 0.0 0.5 1.0 0 5 10 15 20 0 2 4 0 2 4 6 8 10 Ac ti ve hi st o n e ma rk s re p re ss ive hi st o n e ma rk
NB4-LR1
NB4-LR1
SFDuntreated ATRA untreated ATRA ATO ATRA+ATO
H3 (% i nput) H3 K 2 7 M e 3 (% i n put) H3 K 4 M e 3 (% in p u t) H3 A c (% in p u t) 0.1 a b c d e f g h i a b c d e f g h i a b c d e f g h i a b c d e f g h i a b c d e f g h i a b c d e f g h i hi st o n e
1
2
3
4
Fig. 4. Chromatin marks athTERT gene. ChIP was analyzed by quantitative PCR (qPCR) using nine pairs of primers to amplify selected regions (a to i) encompassing thehTERT gene as indicated on the schematic representation above the graphs. Semitransparent gray and pink color labels the analyzed regions ofhTERT proximal promoter and enhancer, respectively. Site-specific ChIPs of H3Ac, H3K4Me3, H3K27Me3, and H3 are presented as percentage of the input the SEM of three independent experiments.
decrease occurred at the hTERT core promoter (region mapped by primers c, d, and e) as well as in the region mapped by the primer set a.
In NB4-LR1 cells, the repressive H3K27Me3 mark was enriched upstream and downstream of the hTERT
core promoter regions (Fig.4, panel 3). Its level
fur-ther increased after ATRA treatment. In contrast, in
NB4-LR1SFD cells, the level of this mark remained
rather weak, even though a modest increase was observed when treated with ATRA and ATO in com-bination. Histone H3 pull-down was used to map the underlying distribution of histones ChIP experiments using anti-histone H3 to map the distribution of his-tones revealed a higher level of histone H3 in
ATRA-treated NB4-LR1 cells than in unATRA-treated ones (Fig.4,
panel 4). Such a difference was not readily observable
in NB4-LR1SFD cells. As mentioned above, this
increase is likely to reflect a change in nucleosome
positioning in this region of the hTERT gene (Fig.3).
Altogether, these results confirm that changes in
hTERT expression are associated with changes in the
pattern of histone post-translational modifications. Furthermore, and importantly, they identify a new
hTERTgene region located at about 5 kb upstream
of the TSS enriched for active H3K4Me3 mark or
H3K27Me3 in NB4-LR1SFD and NB4-LR1 cells,
respectively. The loss of the loss of the H3K4Me3 marks is correlated to hTERT repression.
Next we used publicly available data from three APL patients to investigate whether the primary blasts from these patients present a pattern of histone
modifi-cations similar to NB4-LR1 or NB4-LR1SFDcell lines.
We observed the co-occurrence at hTERT promoter of the two H3K4Me3 and H3K27Me3 marks as in NB4-LR1 cells (Fig. S4). In addition, our analyses showed that the putative hTERT enhancer region is marked by the presence of either H3K4Me3 active marks or H3K27Me3 repressive marks in patients pz-289 and pz-302, respectively, these histone marks being mutu-ally exclusive. Of note, this pattern of histone modifi-cations did not change upon in vitro ATRA treatment. This absence of effect compared to that observed in the APL cell lines can possibly result from the short duration of the treatment, only for 24 h, compared to the 7-day treatment of APL cell lines.
3.5. Analysis of the spatial genome organization
athTERT locus by 3D-FISH
The 3D genome spatial organization has been recently proposed to control nuclear structure and gene expres-sion. Indeed, spatial chromosome arrangement can bring regulatory elements nearby the genes under their
control (Dekker and Mirny, 2016). Likewise, telomeres
can make looping structures and partly regulate gene expression, including the hTERT gene located 1.2 Mb
from the end of chromosome 5p (Kim et al., 2016;
Robin et al., 2014). This mechanism, known as
telom-ere position effect over long distances (TPE-OLD), would possibly influence gene expression over long dis-tances. Based on recent observations supporting the concept that TPE-OLD can induce hTERT repression
(Kim and Shay, 2018), we performed 3D-FISH on
NB4-LR1 and NB4-LR1SFDcell lines with or without
ATRA treatment to evaluate whether potential modifi-cations of spatial genome organization at the hTERT locus would correlate with hTERT expression. We used three BAC probes corresponding to each region of interest: subtelomeric and intermediate sequences,
and TERT locus (Fig.5Aand Fig. S2). An example of
the images acquired is shown in Fig. 5B. We measured
the three-dimensional distances of FISH signals
between the hTERT locus and the 5p subtelomeric region and we discriminated pairs in cis (i.e., on the same chromosome 5) from those in trans (i.e., on dif-ferent chromosomes 5). The spots in a pair present on the same chromosome 5 were then categorized into adjacent, intermediate, and separated. As previously reported, we analyzed the distributions of spots in the two extreme categories, that is, adjacent and separated
(Kim et al., 2016; Kim and Shay,2018). Because
NB4-LR1 cells are slightly smaller than NB4-NB4-LR1SFD cells
thresholds were adapted. Spots were classified as adja-cent when the distance measured was lower than 300
or 340 nm in NB4-LR1 and NB4-LR1SFD cells,
respectively. Spots were classified as separated when the distance was greater than 665 or 730 nm, in
NB4-LR1 and NB4-NB4-LR1SFDcells, respectively (Fig.5C). In
the nucleus of control NB4-LR1 and NB4-LR1SFD
cells, the percentages of spots in close proximity
(adja-cent) were ~ 37% and ~ 43%, respectively. After
7 days of ATRA treatment, this percentage was
signifi-cantly increased to ~ 55% in the nucleus of NB4-LR1
cells, while the separated pairs decreased from ~ 63%
to ~ 45%. In contrast, the distances between probes
corresponding to these loci remained unchanged in the
nucleus of ATRA-treated NB4-LR1SFD cells as
com-pared to untreated cells (Fig. 5C). Of note, we did not
observe any differences in the distances between probes corresponding to the hTERT locus and the ‘in-termediate’ sequences between untreated and
ATRA-treated in both NB4-LR1 and NB4-LR1SFD cells
(Fig. 5D). Overall, these results suggest that, in
NB4-LR1 cells, hTERT repression by ATRA treatment could be modulated by long-range TPE-OLD interac-tions at the hTERT locus.
4. Discussion
Since hTERT expression is upregulated in the majority of tumors, the elucidation of the mechanisms responsi-ble for hTERT regulation will offer information that may be used for diagnosis and prognosis and could be translated into effective targeted cancer therapies. DNA methylation is functionally linked to several other epige-netic pathways, including post-translational histone
modifications (Okitsu and Hsieh, 2007; Weber et al.,
2007), nucleosome positioning (Huff and Zilberman,
2014), and nuclear localization. As these processes play
an essential role in gene expression by regulating the condensation and accessibility of genomic DNA, they were investigated at the level of hTERT gene to obtain a comprehensive view of the epigenetic landscape of this gene and consequently its regulation. Therefore, we investigated the epigenetic landscape changes associated with ATRA-induced hTERT gene repression with a unique approach combining several techniques.
4.1. DistincthTERT promoter DNA methylation
patterns identified in APL cell lines and patients Driving mutations in hTERT promoter have been found
in over 50 cancer types (Vinagre et al.,2013). Although
these mutations are frequent in certain types of cancers, they are rarely found in other types, including breast, prostate, lung cancers, and leukemia. In the absence of
hTERTmutations, the sustained expression and
overex-pression of hTERT in cancer cells may occur through an epigenetic switch. It can be hypothesized that this switch mechanism mimics the consequences of promoter muta-tions. It has been well documented that DNA methyla-tion, histone acetylamethyla-tion, and methylation are involved in the regulation of hTERT transcription (Leao et al.,
2018; Yuan and Xu, 2019). However, the role and the
molecular mechanisms are not well depicted and can be even contradictory due to the different cellular models, the different areas within the hTERT promoter studied, and the various methods used to analyze these epige-netic modifications. Furthermore, understanding the epigenetic regulation of hTERT requires a global analy-sis of the relationship between DNA methylation, his-tone modifications, and nucleosome positioning.
Our previous study performed on APL cell lines iden-tified two distinct functional domains in hTERT pro-moter, one distal and one proximal, differentially
methylated (Azouz et al., 2010). These results are in
agreement with the recent identification of the TERT hypermethylation oncologic region, a 433-bp genomic
region located159 to 591 bp upstream the TSS
(Cas-telo-Branco et al., 2016; Leao et al., 2019; Lee et al.,
2019), as a potential biomarker in several cancers. This
region is of major importance, because its methylation status correlates with hTERT expression. A similar pro-file has also been reported in other cancer cells
express-ing telomerase (Zinn et al.,2007). In the present study,
we depicted similar functional regions of the hTERT core promoter in APL patients at diagnosis. Upon dis-ease remission, all APL patients present a decrdis-eased DNA methylation in the distal domain of the hTERT core promoter, as compared to patient at diagnosis, leading to a pattern of methylation similar to that in healthy individuals. In vivo, ATRA treatment causes the abnormal promyelocytes to differentiate into mature leukocytes; however, their clearance is not known. The results obtained in patients favor the hypothesis that the difference in the methylation pattern at the hTERT core promoter between patients at diagnosis and patients at remission after ATRA treatment is not due to a change of pattern during differentiation of abnormal promyelo-cytes but rather to a disappearance of predominant abnormal promyelocytes in favor of normal promyelo-cytes. This observation on patient specimen is in perfect agreement with the results obtained in NB4-LR1 cells showing that long-term ATRA treatment induced a decrease of DNA methylation in the same region, which is accompanied by hTERT repression.
Altogether, this finding, however, reinforces the idea that DNA methylation pattern of this region represents a potential indicator for monitoring the disease out-come. Furthermore, it raises the question of whether targeting the methylation status of this distal region of
hTERTcore promoter may have a therapeutic interest.
We used nucleosome occupancy and methylation sequencing (NOMe-seq) assay for the first time to mea-sure simultaneously endogenous DNA methylation and nucleosome occupancy at hTERT gene promoter. The low level of methylation at hTERT TSS was similar between cells that repressed hTERT (ATRA-treated
NB4-LR1 and ATRA+ ATO-treated NB4-LR1SFD
cells) and cells that maintained hTERT expression
(un-treated NB4-LR1 and NB4-LR1SFD cells, ATRA- or
ATO-treated NB4-LR1SFD cells). Despite the constant
low level of methylation in the proximal part of hTERT promoter, modifications in chromatin accessibility were observed correlated with a reduction of active histone marks and hTERT transcriptional activity. Interest-ingly, our study shows that some sites of differential accessibility did not show the expected differential DNA methylation pattern indicating that, as already reported
(Collings and Anderson,2017), some epigenomic
chro-matin components other than DNA methylation, including histone modifications, are responsible for dif-ferential chromatin accessibility.
4.2. Identification of specific histone
modification marks associated withhTERT
regulation by retinoids
We showed in NB4-LR1 cells that hTERT promoter has the features of a bivalent promoter, that is, the
simultaneous presence of the repressive mark
H3K27Me3 and the activation mark H3K4Me3
around the TSS. These marks are commonly seen on bivalent genes ‘poised’ for activation upon stem cell commitment and differentiation. In embryonic stem cells, bivalent promoters may achieve the possibility to respond rapidly to incoming signals by suppressing the formation of active RNA polymerase II complexes on
Ch r 5 p hTERT probe (RP11-117B23) (160 kb) Subtelomeric probe (RP11-44H14) (180 kb) Intermediate probe (RP11-846K3) (100 kb) Telomere Centromere 0.6 Mb 0.6 Mb 0.2 Mb NB4-LR1SFD Control ATRA ns 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 D ist anc e (nm) NB4-LR1
**
0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 Di st an ce ( n m ) hT ER T-sub tel omeri c pro b es Control 42.6 57.4 Adjacent <340 nm Separated >730 nm 46.1 53.9 36.7 63.3 Adjacent <300 nm Separated >665 nm 55.5 44.5 ATRA A B C D Adjacent <230 nm Separated >525 nm 40.3 59.7 41.5 58.5 Adjacent <250 nm Separated >560 nm 45.2 54.8 39.8 60.2 0 5 0 0 1 0 0 0 1 5 0 0 control ATRA ns NB4-LR1SFD D ist anc e (nm) NB4-LR1 ns control ATRA 0 5 0 0 1 0 0 0 1 5 0 0 D ist anc e (nm) hT ER T-in te rm edi at e pro b es TERT 44H14 2µm 2µm 846K3 2µm TERTFig. 5. 3D DNA-FISH analysis. (A) The location of each probe is shown in a schematic representation of the short arm (p) of chromosome 5. DNA probes (RP11-117B23 BAC) againsthTERT locus (red) and targeting subtelomeric 5p region (RP11-44H14 BAC, green) are used to analyze physical proximity by 3D-FISH. (B) Confocal images of 3D-FISH afterIMAGEJprocessing. Representative confocal images of 3D-FISH showing either the proximity or the separation of the loci. DNA was counterstained with Hoechst. Arrow heads indicate spots that are considered adjacent; full arrows indicate spots that are separated. (C, D) Box plots (upper panels) illustrate the distribution of the distances determined from processed images ofin situ hybridization using either the subtelomeric (C) or the intermediate probe (D) relative to the hTERT probe in NB4-LR1 variants before and after ATRA treatment (1 µM, 7 days). Images were processed withIMAGEJfor about 150–200
nuclei from three separate experiments per condition. Significant differences are marked with asterisks (Mann–Whitney test). Pie charts (lower panels) illustrate the relative proportion of adjacent and separated signals in NB4-LR1 and NB4-LR1SFDcells. The criteria for the
analyses depending on both the probes and the cell lines are indicated. BAC probes were more adjacent in the ATRA-treated NB4-LR1 cells than in control cells.
the one hand, and, on the other hand, not allow other less reversible suppressive regulatory mechanisms, like DNA methylation, to silence genes. This feature demonstrates the plasticity of the hTERT gene in NB4-LR1 cell variants contributing to its reactivation during cellular transformation and tumorigenesis in a permissive environment. Indeed, ATRA treatment induced a strong decrease of the active histone marks, whereas enrichment of repressive marks was observed.
By contrast, in NB4-LR1SFD cells, the levels of
H3K27Me3 were significantly lower. In this cell line, only the ATRA and ATO combined treatment induced a slight enrichment of this repressive mark associated with a decrease of the active histone marks more important for H3Ac than H3K4Me3.
Importantly, this study points to a role of a new region in activating hTERT. This region is localized outside the minimal promoter approximately 5 kb
upstream of the TSS and has been previously
described as an enhancer (Eldholm et al., 2014). This
domain is differentially methylated in both cell lines,
being hypermethylated in NB4-LR1 cells and
hypomethylated in NB4-LR1SFD cells. Interestingly
and in contrast with NB4-LR1 cells, in NB4-LR1SFD
cells, this region of hTERT promoter was enriched in both H3Ac and H3K4Me3 active marks. This feature has also been observed in one of the APL patient sam-ples analyzed in this study. H3K4Me3 is a predomi-nant feature of active promoters, but detectable levels of this modification are also observed at active
enhan-cers (Pekowska et al., 2011). Compared to NB4-LR1
cells, a high level of H3K4Me3 was observed in the enhancer domain of hTERT in the untreated
NB4-LR1SFD cells. Although the mechanisms underlying
this difference in H3K4Me3 deposition at hTERT enhancer remain to be further explored, one potential explanation is that it may reflect a difference in the DNA methylation pattern of this region. Indeed, trimethylation of H3K4 (H3K4Me3) is mostly per-formed by the CxxC finger protein 1 (Cfp1), a subunit of the human Set1 complex, which influence chromatin structure through its binding to unmethylated CpGs and links H3K4Me3 with CpG islands (Lee and
Skal-nik, 2005; Thomson et al., 2010). However, Cpf1 also
plays a role in the production of H3K4Me3 at other regulatory regions, including distal enhancers (Clouaire
et al.,2012; van de Lagemaat et al., 2018). As in
NB4-LR1SFD cells, the hTERT enhancer region is mainly
hypomethylated compared to NB4-LR1 cells, it could be targeted by Cfp1 (or other CpG-binding proteins) leading to the ectopic deposition of H3K4Me3 and to an aberrantly elevated level of transcription both from the enhancer and from the nearby promoter. This
feature can explain the higher constitutive expression
of hTERT in NB4-LR1SFD cells than in NB4-LR1
cells. In contrast in NB4-LR1 cells, densely methylated CpGs in the hTERT enhancer region are likely to
attract methyl-CpG-binding proteins that recruit
enzymes reinforcing repressive histone modifications (i.e., H3K27Me3).
In NB4-LR1SFDcells, only a combination of ATRA
and ATO treatments induced hTERT repression asso-ciated with a drastic drop of the levels of H3K4Me3 and H3Ac marks at hTERT enhancer and promoter. In NB4-LR1 cells, the hTERT enhancer is not active and consequently ATRA alone was able to induce
hTERT repression provided both the enhancer and
promoter region of hTERT have been enriched with the H3K27Me3 repressive mark and hTERT promoter depleted of the and H3K4Me3/H3Ac active marks
(Fig.6).
Altogether, these results suggest that the epigenetic features of this region could play a major role and dic-tate the context-dependent hTERT transcriptional out-come, through differential recruitment of transcription factors and other chromatin-modifying enzymes. In
sil-icoanalysis of this region identifies putative consensus
binding sites for multiple interacting transcription
fac-tors, including RARa/RXRc, PPARa/RXRa, Sp1,
and GABPa. Factors, such as CTCF, that interact
with hTERT promoter (either proximal promoter or enhancer) are known for organizing global chromoso-mal architecture, changing accessibility, but also possi-ble interactions far away in distance. Thus, it can be proposed that a potential cross-talk exists between the enhancer and the core promoter of hTERT gene to orchestrate its expression. As transcription factors could interact with some epigenetic modifiers, a better understanding of the cooperation between transcrip-tion factors and the epigenetic landscape will offer hope to develop new drug opportunities. The epige-netic modifications reported above could partially par-ticipate in regulation of hTERT gene expression via telomere looping in a context not related to telomere length modifications as suggested in the present study
and also recently reported (Kim and Shay,2018).
Retinoid-based therapies have low systemic toxicity when compared to conventional chemotherapeutics. Recent studies indicate that ATRA-based therapy may be of benefit to patients with other cancers. However, the individuality in the clinical response shows clearly that a treatment protocol may not be effective in all cases. The present work identifies some specific fea-tures of hTERT epigenetic landscape that could be used to predict the response of patients to ATRA-based therapy.
5. Conclusions
Together, our results suggest that the local chromatin accessibility of the core promoter of hTERT gene is likely the most important feature controlling the tran-scriptional expression of hTERT gene. However, it could not be excluded that the methylation pattern of the enhancer domain of hTERT gene is associated with specific histone modifications that could play a role in
hTERTactivation.
Therefore, our results suggest a very complex rela-tionship between the epigenetic state of hTERT pro-moter and transcriptional activity and, thereby, force the revisiting of some previously proposed concepts regarding hTERT regulation. The analysis of the epi-genetic status of hTERT as performed in this study can provide the basis for further works to extend these findings and translate them into promising new
approaches for the treatment of a broad range of can-cers. As next generation sequencing technologies asso-ciated with new bioinformatics techniques and analysis tools are rapidly evolving, new opportunities are pro-vided to identify epigenetic landscape changes that can be successfully and reliably used in clinical practice to follow the disease and the response to treatment.
Acknowledgements
We acknowledge the Cyto2BM and the Imaging and Microscopy (SCM) core facilities of BioMedTech
Facilities INSERM US36, CNRS UMS2009,
Univer-site de Paris, France. The work of the authors was supported by grants from the Institute of Health and Medical Research (INSERM), the National Center for Scientific Research (CNRS), the Fondation de France (n° 201300038226 to ES-B), the French National
100% DNA methylation H3Ac Histone modifications 40–50% 0–10% H3K4Me3 H3K27Me3 Nucleosomes Ac tor Repressor Transcription factors R A Transcripon A A Corepromoter Transcription A A A hTERT Enhancer Repression R hTERT ATRA B NB4-LR1SFD Transcription A R A A hTERT ATO ATRA +ATO Enhancer Corepromoter Transcription A A hTERT Repression R hTERT ATRA hTERT A NB4-LR1 proximal distal proximal distal
Fig. 6. Proposed model of epigenetic regulation ofhTERT expression. hTERT promoter and enhancer exhibit distinct epigenetic features in NB4-LR1 (A) and NB4-LR1SFD(B) cells. (A) In NB4-LR1 cells, under ATRA treatment, the repressive histone mark (H3K27Me3) is enriched at
the hTERT core promoter whereas the active marks (H3Ac and H3K4Me3) are depleted. These modifications are associated with an increase in the number of nucleosomes, a decrease in chromatin accessibility, andhTERT repression. In addition, the DNA methylation of the distal part ofhTERT core promoter decreases. This hypomethylation pattern would possibly allow the binding of a repressive factor (R) partly responsible for ATRA-inducedhTERT repression. The enhancer domain of hTERT remains unmodified. (B) In NB4-LR1SFDcells, the
enhancer domain is characterized by active histone marks and hypomethylated DNA. This decrease would possibly allow the binding of an activator (A).hTERT is repressed only after treatment with ATRA and ATO in combination. This hTERT repression is associated, as in NB4-LR1 cells, with a demethylation of the distalhTERT promoter and a condensation of the chromatin in the proximal part of hTERT promoter. Moreover, in contrast with NB4-LR1, in NB4-LR1SFD cells, hTERT repression involves the inactivation of the enhancer region of hTERT characterized by a decrease of the histone active marks. ATRA treatment alone does not induce any modification of DNA methylation, histone modifications, and chromatin accessibility in the hTERT core promoter. However, a weak but significant increase of DNA methylation in the enhancer region could partly explain the initialhTERT repression observed after ATRA treatment of the NB4-LR1SFDcells.
ATO treatment alone does not change DNA methylation of this enhancer region. However, it decreases the DNA methylation of the distal hTERT promoter. As mentioned above for NB4-LR1 cells, this can favor the binding of a repressor and explain partly the initial repression of hTERT observed after ATO treatment. From this model we extrapolate that an inhibition of the enhancer activity associated with epigenetic modifications at both distal and proximal domain ofhTERT promoter are necessary for a full repression of hTERT promoter.
Research Agency (ANR to CB), Ministere de l’Enseignement Superieur, de la Recherche et de l’Innovation (MESRI to DG), the Ligue nationale contre le cancer (Comite d’Ile de France, ES-B), the French Society of Dermatology (MP-C and EC), and Charpak Lab scholarship (RK).
Conflict of interest
The authors declare no conflict of interest.
Author contributions
DG, CB, and EN performed the experimental work. RK performed the APL patient dataset analysis. DG, EN, RK, and ES-B analyzed the data. MP-C and EC contributed biological materials. DG, CB, EN, MP-C, EC, and PF contributed to the drafted manuscript and to its critical revision. ES-B designed the study and wrote the manuscript. The final manuscript was read and approved by all authors. All authors discussed the results and commented on the manuscript.
Data availability
The APL dataset analyzed in the current study is available at the Gene Expression Omnibus (https://
www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=
GSE42119) and at the BLUEPRINT data portal (http://dcc.blueprint-epigenome.eu/#/experiment).
References
Avilion AA, Piatyszek MA, Gupta J, Shay JW, Bacchetti S and Greider CW (1996) Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Can Res56, 645–650.
Azouz A, Wu YL, Hillion J, Tarkanyi I, Karniguian A, Aradi J, Lanotte M, Chen GQ, Chehna M and Segal-Bendirdjian E (2010) Epigenetic plasticity of hTERT gene promoter determines retinoid capacity to repress telomerase in maturation-resistant acute promyelocytic leukemia cells. Leukemia24, 613–622.
Bannister AJ and Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res21, 381– 395.
Bell RJ, Rube HT, Kreig A, Mancini A, Fouse SD, Nagarajan RP, Choi S, Hong C, He D, Pekmezci M et al. (2015) Cancer. The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer. Science348, 1036–1039. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert
DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K
et al. (2006) A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell125, 315–326.
Castelo-Branco P, Leao R, Lipman T, Campbell B, Lee D, Price A, Zhang C, Heidari A, Stephens D, Boerno S et al. (2016) A cancer specific hypermethylation signature of the TERT promoter predicts biochemical relapse in prostate cancer: a retrospective cohort study. Oncotarget7, 57726–57736.
Chaumeil J, Micsinai M and Skok JA (2013) Combined immunofluorescence and DNA FISH on 3D-preserved interphase nuclei to study changes in 3D nuclear organization. J Vis Exp72, e50087.
Chodavarapu RK, Feng S, Bernatavichute YV, Chen PY, Stroud H, Yu Y, Hetzel JA, Kuo F, Kim J, Cokus SJ et al. (2010) Relationship between nucleosome positioning and DNA methylation. Nature466, 388– 392.
Clouaire T, Webb S, Skene P, Illingworth R, Kerr A, Andrews R, Lee JH, Skalnik D and Bird A (2012) Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells. Genes Dev26, 1714–1728.
Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ and Reddel RR (2007) Protein composition of catalytically active human telomerase from immortal cells. Science315, 1850–1853.
Collings CK and Anderson JN (2017) Links between DNA methylation and nucleosome occupancy in the human genome. Epigenetics Chromatin10, 18.
Cong YS and Bacchetti S (2000) Histone deacetylation is involved in the transcriptional repression of hTERT in normal human cells. J Biol Chem275, 35665–35668. Dekker J and Mirny L (2016) The 3D genome as
moderator of chromosomal communication. Cell164, 1110–1121.
Dessain SK, Yu H, Reddel RR, Beijersbergen RL and Weinberg RA (2000) Methylation of the human telomerase gene CpG island. Can Res60, 537–541. Devereux TR, Horikawa I, Anna CH, Annab LA, Afshari
CA and Barrett JC (1999) DNA methylation analysis of the promoter region of the human telomerase reverse transcriptase (hTERT) gene. Can Res59, 6087– 6090.
Duprez E, Ruchaud S, Houge G, Martin-Thouvenin V, Valensi F, Kastner P, Berger R and Lanotte M (1992) A retinoid acid ‘resistant’ t(15;17) acute promyelocytic leukemia cell line: isolation, morphological,
immunological, and molecular features. Leukemia6, 1281–1287.
Eldholm V, Haugen A and Zienolddiny S (2014) CTCF mediates the TERT enhancer-promoter interactions in lung cancer cells: identification of a novel enhancer region involved in the regulation of TERT gene. Int J Cancer134, 2305–2313.
Guilleret I and Benhattar J (2003) Demethylation of the human telomerase catalytic subunit (hTERT) gene promoter reduced hTERT expression and telomerase activity and shortened telomeres. Exp Cell Res289, 326–334.
Guilleret I, Yan P, Grange F, Braunschweig R, Bosman FT and Benhattar J (2002) Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity. Int J Cancer101, 335–341.
Hanahan D and Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell144, 646–674.
Horn S, Figl A, Rachakonda PS, Fischer C, Sucker A, Gast A, Kadel S, Moll I, Nagore E, Hemminki K et al. (2013) TERT promoter mutations in familial and sporadic melanoma. Science339, 959–961.
Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L and Garraway LA (2013) Highly recurrent TERT promoter mutations in human melanoma. Science339, 957–959. Huff JT and Zilberman D (2014) Dnmt1-independent CG methylation contributes to nucleosome positioning in diverse eukaryotes. Cell156, 1286–1297.
Kelly TK, Liu Y, Lay FD, Liang G, Berman BP and Jones PA (2012) Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res22, 2497–2506.
Kim W, Ludlow AT, Min J, Robin JD, Stadler G, Mender I, Lai TP, Zhang N, Wright WE and Shay JW (2016) Regulation of the human telomerase gene TERT by telomere position effect-over long distances (TPE-OLD): implications for aging and cancer. PLoS Biol 14, e2000016.
Kim W and Shay JW (2018) Long-range telomere regulation of gene expression: telomere looping and telomere position effect over long distances (TPE-OLD). Differentiation99, 1–9.
Lai WKM and Pugh BF (2017) Understanding nucleosome dynamics and their links to gene expression and DNA replication. Nat Rev Mol Cell Biol18, 548–562. Lanotte M, Martin-Thouvenin V, Najman S, Balerini P,
Valensi F and Berger R (1991) NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood77, 1080–1086.
Leao R, Apolonio JD, Lee D, Figueiredo A, Tabori U and Castelo-Branco P (2018) Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer. J Biomed Sci25, 22. Leao R, Lee D, Figueiredo A, Hermanns T, Wild P,
Komosa M, Lau I, Mistry M, Nunes NM, Price AJ et al. (2019) Combined genetic and epigenetic alterations of the TERT promoter affect clinical and biological behavior of bladder cancer. Int J Cancer 144, 1676–1684.
Lee DD, Leao R, Komosa M, Gallo M, Zhang CH, Lipman T, Remke M, Heidari A, Nunes NM, Apolonio JD et al. (2019) DNA hypermethylation within TERT promoter upregulates TERT expression in cancer. J Clin Investig129, 223–229.
Lee JH and Skalnik DG (2005) CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex. J Biol Chem280, 41725–41731.
Lewis KA and Tollefsbol TO (2016) Regulation of the telomerase reverse transcriptase subunit through epigenetic mechanisms. Front Genet7, 83. Li B, Carey M and Workman JL (2007) The role of
chromatin during transcription. Cell128, 707–719. Losi L, Lauriola A, Tazzioli E, Gozzi G, Scurani L,
D’Arca D and Benhattar J (2019) Involvement of epigenetic modification of TERT promoter in response to all-trans retinoic acid in ovarian cancer cell lines. J Ovarian Res12, 62.
Mallona I, Diez-Villanueva A and Peinado MA (2014) Methylation plotter: a web tool for dynamic visualization of DNA methylation data. Source Code Biol Med9, 11.
Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, Ziaugra L, Beijersbergen RL, Davidoff MJ, Liu Q et al. (1997) hEST2, the putative human telomerase catalytic subunit gene, is up- regulated in tumor cells and during immortalization. Cell90, 785– 795.
Okitsu CY and Hsieh CL (2007) DNA methylation dictates histone H3K4 methylation. Mol Cell Biol27, 2746– 2757.
Pekowska A, Benoukraf T, Zacarias-Cabeza J, Belhocine M, Koch F, Holota H, Imbert J, Andrau JC, Ferrier P and Spicuglia S (2011) H3K4 tri-methylation provides an epigenetic signature of active enhancers. EMBO J 30, 4198–4210.
Pendino F, Dudognon C, Delhommeau F, Sahraoui T, Flexor M, Bennaceur-Griscelli A, Lanotte M and Segal-Bendirdjian E (2003) Retinoic acid receptor alpha and retinoid-X receptor-specific agonists synergistically target telomerase expression and induce tumor cell death. Oncogene22, 9142–9150.
Pendino F, Flexor M, Delhommeau F, Buet D, Lanotte M and Segal-Bendirdjian E (2001) Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation. Proc Natl Acad Sci USA98, 6662–6667.
Pendino F, Hillion J, Dudognon C, Delaunay J, Mourah S, Podgorniak MP, Lafon I, Chomienne C, Lanotte M, Dombret H et al. (2006) Telomerase targeting by retinoids in cells from patients with myeloid leukemias of various subtypes, not only APL. Leukemia20, 599– 603.
